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Oncotarget1www.impactjournals.com/oncotarget
Inhibition of the PI3K/AKT/mTOR pathway activates autophagy and compensatory Ras/Raf/MEK/ERK signalling in prostate cancer
Dominika E. Butler1, Christopher Marlein2, Hannah F. Walker1, Fiona M. Frame1,
Vincent M. Mann3, Matthew S. Simms4,5, Barry R. Davies6, Anne T. Collins1 and
Norman J. Maitland1
1The Cancer Research Unit, Department of Biology, University of York, York YO10 5DD, UK
2Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK
3Gastroenterology Research Department, 8th Floor Alderson House, Hull Royal Inirmary, Anlaby Road, Hull, HU3 2JZ, East Riding of Yorkshire, UK
4Department of Urology, Castle Hill Hospital (Hull & East Yorkshire Hospital NHS Trust), Cottingham, HU16 5JQ, UK
5Hull York Medical School, University of York, YO10 5DD, UK
6AstraZeneca, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE
Correspondence to: Norman J. Maitland, email: [email protected]
Keywords: prostate cancer, AKT, mTOR, RAS, MAPK signalling, autophagy
Received: May 27, 2016 Accepted: April 24, 2017 Published: May 23, 2017
Copyright: Butler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
The PI3K/AKT/mTOR pathway is frequently activated in advanced prostate
cancer, due to loss of the tumour suppressor PTEN, and is an important axis for drug
development. We have assessed the molecular and functional consequences of pathway
blockade by inhibiting AKT and mTOR kinases either in combination or as individual
drug treatments. In established prostate cancer cell lines, a decrease in cell viability
and in phospho-biomarker expression was observed. Although apoptosis was not
induced, a G1 growth arrest was observed in PTEN null LNCaP cells, but not in BPH1 or
PC3 cells. In contrast, when the AKT inhibitor AZD7328 was applied to patient-derived
prostate cultures that retained expression of PTEN, activation of a compensatory Ras/
MEK/ERK pathway was observed. Moreover, whilst autophagy was induced following
treatment with AZD7328, cell viability was less affected in the patient-derived cultures
than in cell lines. Surprisingly, treatment with a combination of both AZD7328 and two
separate MEK1/2 inhibitors further enhanced phosphorylation of ERK1/2 in primary
prostate cultures. However, it also induced irreversible growth arrest and senescence.
Ex vivo treatment of a patient-derived xenograft (PDX) of prostate cancer with
a combination of AZD7328 and the mTOR inhibitor KU-0063794, signiicantly reduced tumour frequency upon re-engraftment of tumour cells.
The results demonstrate that single agent targeting of the PI3K/AKT/mTOR
pathway triggers activation of the Ras/MEK/ERK compensatory pathway in near-
patient samples. Therefore, blockade of one pathway is insuficient to treat prostate cancer in man.
INTRODUCTION
Prostate cancer is the most common cancer in men,
and the second most common cause of cancer death in
men in the UK (Cancer Research UK, 2014). Advanced
prostate tumours initially respond well to androgen
deprivation therapy (ADT). However, in most cases, ADT
ultimately fails and castration resistant prostate cancer
(CRPC) emerges, which is almost inevitably fatal [1].
Although novel therapeutic agents, such as abiraterone
www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2017
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and enzalutamide have demonstrated improved overall
survival in metastatic CRPC, new therapies are urgently
needed [2�4]. There is also a growing realisation that early
adoption of chemotherapy either before or with hormone
therapy, results in greatly increased survival [5].
The PI3K/AKT/mTOR pathway is over-activated
in many human cancers, including prostate cancer, and
has emerged as a target for small molecule therapies
[6�8]. However, inhibition of the PI3K pathway alone
has proven to be insuficient for treatment of cancer, due to the existence of feedback loops and cross-talk
with other pathways, including the Ras/Raf/MEK/ERK
[9�11], and androgen receptor signalling pathways [8,
12, 13]. Therefore, combined targeting of the PI3K/
AKT/mTOR and the Ras/MEK/ERK pathways has
been proposed as an alternative approach in a range
of different cancers, including multiple myeloma
(MM), melanoma and prostate cancer [6, 14, 15]. In
multiple myeloma, combined blockade of the PI3K and
MAPK pathways resulted in signiicantly enhanced cell death in the majority of tested cell lines [14]. A
combination of the mTORC1 inhibitor, rapamycin and
the MEK inhibitor, PD325901 inhibited growth of both
androgen-responsive (CWR22Rv1 and CASP 2.1) and
androgen-independent (CASP 1.1) prostate cancer
cell lines. Blockade of both pathways, using a novel
sorafenib derivative, NSK-01105, was shown to reduce
cell proliferation and induce apoptosis in LNCaP and
PC3 cells, and xenografted tumours of the same cell
lines [16].
Preclinical assessment of potential new prostate
cancer therapies is routinely performed in a limited
number of cell lines and xenografts derived from them.
The acknowledged heterogeneity between responses
in different patients is therefore not addressed and may
be the cause of the high attrition rate in oncology drug
development [17]. In general, such approaches allow
measurements of drug eficacy, but can prove misleading in determining the clinical applicability of drugs and drug
combinations. Additionally, the presence of cancer stem
cells (CSCs), in a range of cancers including leukaemia
[18], breast [19], brain [20], colon [21], pancreas [22],
lung [23] and prostate [24�26], has been suggested to be
responsible for therapy resistance [27]. In prostate cancer,
basal phenotype CSCs, CD44+/g2く
1
hi/CD133+ are the
most likely source of treatment-resistant cells, serving as
a reservoir for tumour recurrence after castration therapy
[24, 28, 29].
To address intra-tumour heterogeneity and differ-
ences between patients, we investigated the role of PI3K/
AKT/mTOR signalling in a range of prostate cancer
primary cultures and PDXs. By measuring the signalling
and cellular responses after treatment with small molecule
inhibitors of the pathway, we demonstrate a surprising,
but consistent disparity between the �standard� cell line
models and patient-derived cells.
RESULTS
Novel AKT and mTOR inhibitors reduce
prostate cell viability
As AKT and mTOR kinases are crucial regulators
of prostate cancer cell growth, proliferation and survival,
treatment of high grade tumours with an AKT kinase
inhibitor (AZD7328) and mTOR kinase inhibitor (KU-
0063794), both ATP-competitive small molecules, has
been proposed as a promising treatment strategy in
oncology [6–8]. To establish the eficacy of the small molecule inhibitors, two PTEN-positive cell lines, BPH1
(benign) and P4E6 (localised cancer) and two PTEN-
negative cell lines LNCaP (lymph node metastasis)
and PC3 (bone metastasis) were treated with a range
of concentrations (0.01 – 30 たM) of AZD7328 or KU-0063794, for up to 48 hours. An MTS assay was performed
to investigate the effect of AZD7328 and KU-0063794 on
cell viability. LNCaP cells were the most sensitive to both
inhibitors with an EC50
of 0.4 たM (0.3-0.5) for AZD7328 and 1.03 たM (0.8-1.3) for KU-0063794 (Table 1). PC3 cells were similarly susceptible to KU-0063794, but were
27 fold less sensitive to AZD7328 (11.0 たM). Although treatment with AZD7328 reduced the viability of both
PTEN-negative cell lines, the PTEN-positive cells lines
(BPH1 and P4E6) were less sensitive to the AKT inhibitor
and their viability was only slightly reduced. Speciically, the EC
50 for the KU-0063794 were approximately
5 – 15 fold higher for BPH1 (5.8 たM) and P4E6 (10.2 たM) in comparison to LNCaP and PC3 (Table 1). Taken together, these results suggest that AZD7328 and KU-
0063794�s ability to reduce cell viability is dependent on
PTEN status, with PTEN-negative cell lines being more
susceptible to AKT and mTOR blockade.
To validate these indings in a ‘near-patient’ setting, primary cultures were generated from prostate cancer
biopsies. Biopsies from patients with benign disease
were used as normal controls. The cells were treated
with increasing concentrations (1 � 50 µM) of AZD7328
or KU-0063794 for up to 72 hours and cell viability
was assessed. The results show that AZD7328 and KU-
0063794 are less effective in patient-derived cultures
(Supplementary Figure 1) indicating that combined
treatment should be considered.
As mTOR kinase is responsible for maintaining
metabolic homeostasis, the effect of treatment was
measured using trypan blue exclusion to assess viability,
and in a larger cohort of patients (n=6). Primary cultures
were treated with AZD7328 and KU-0063794, either
alone or in combination. We observed a signiicant decrease in the viability (~ 30%) with 3 たM KU-0063794 treatment at 24 and at 48 hours (P< 0.015) (Figure 1A).
Interestingly, a signiicant decrease in cell viability was observed in the majority of samples following combined
treatment with 3 たM of either inhibitor at 24 hours and 48
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hours (P < 0.006) (Figure 1A). Additionally, a signiicant advantage of combination treatment over AZD7328 alone
was observed at 48 hours. Importantly, these data show
that there is variability in response to treatment between
patients.
Since prostate epithelial cells are organised in
a hierarchy [24, 29�31], the effect of treatment was
assessed on the viability of Stem-like Cells (SC), Transit
Amplifying (TA) and Committed Basal (CB) cells.
Primary epithelial cancer cultures (derived from patients
with Gleason 9, n=3 and Gleason 7 cancer, n=1) were
treated with either 3 たM AZD7328, 3 たM KU-0063794 or a combination of 3 たM AZD7328 + 3 たM KU-0063794, for 72 hours. The cell subpopulations were isolated
following treatment, and viability was assessed using
trypan blue exclusion. Treatment with 3 たM AZD7328 reduced the viability of stem-like cells in 3 of 4 samples
with the highest reduction (78%), in a Gleason 9 sample
(H149/12) (Figure 1B). Moreover, treatment with 3 たM KU-0063794 resulted in a decrease in the viability of
SCs derived from all Gleason 9 samples, whilst stem
cell numbers were not affected by treatment from the
Gleason 7 culture. Interestingly, a combination of 3 たM AZD7328 + 3 たM KU-0063794 resulted in a signiicant decrease (P = 0.0044) in SC numbers in all cancer cultures
(Figure 1B).
In contrast to the effect on stem cell numbers,
TA and CB cell numbers were not affected with either
inhibitor alone or a combination of both (Figure 1B). These
results relect prostate cancer intra tumoural heterogeneity as well as differences between patient samples further
emphasizing the need for patient stratiication.
Inhibition of AKT and mTOR does not induce
cell cycle arrest in primary cultures
To determine whether the inhibitors directly
inluenced the cell cycle, two BPH and three cancer cultures were treated for 72 hours, with 5 たM AZD7328, 5 たM KU-0063794, and a combination of 5 たM AZD7328 + 5 たM KU-0063794 and were subsequently analysed by low cytometry. The results revealed no signiicant difference in cell cycle distribution (p value >0.3) (Figure 1C).
Expression of the tumour suppressor PTEN in
prostate cancer patient cultures
Loss of the tumour suppressor gene, PTEN is
observed in 40% of advanced prostate cancers [32, 33].
Given the importance of PTEN in regulating the activity of
the PI3K pathway, we determined the status of the PTEN
gene as well as expression, in our patient-derived cultures.
DNA analysis was performed on 7 samples for which
matched lymphocytes and tumour biopsies were available.
As a mutational hot spot has been reported in exon 5 of
the PTEN gene (http://atlasgeneticsoncology.org/Genes/
PTENID158.html), we focused our analysis on exon 5.
The results showed that missense mutations were found
in tumour DNA of three samples (H135/11, H224/12 and
H278/13), but not in their corresponding lymphocytes
(Supplementary Figure 2A). However, it is not clear
whether those mutations have resulted in translation of
a functional PTEN and be affecting functionality of the
PTEN protein and this will need to be investigated further.
Next, we determined whether tumour cells showed
loss of heterozygosity at the PTEN gene locus 10p23
using two LOH probes (Table 2) described in Dahia et
al [34]. LOH analysis using Probe 1 (dinucleotide repeat
in PTEN intron 2 (AFMa086wg9) showed that 2/7
samples (H135/11 and H377/13) were heterozygous and
both cultures showed a relative loss of heterozygosity
(expressed as reduction in peak intensity) in tumour
DNA. Interestingly, the H135/11 sample which Had
a missense mutation in exon 5 and LOH, showed a
signiicant reduction in cell viability of stem-like cells following treatment with a combination of AZD7328 and
KU-0063794, indicating higher susceptibility to the drugs.
LOH analysis using probe 2 (G/T sequence polymorphism
in PTEN intron 8) identiied heterozygosity in a further 2 samples (H224/12 and H237/12). However, only H224/12
showed a relative loss of heterozygosity in the tumour
DNA in comparison to the lymphocyte DNA.
Western blotting results showed that PTEN protein was
detected in all primary cultures tested (Supplementary Figure
2B). The presence of PTEN in our cultures could possibly
explain the limited eficacy of the AZD7328 and KU-0063794 in comparison to PTEN-null cell lines, such as LNCaP.
Table 1. EC50
for AZD7328 and KU-0063794 in prostate cell lines.
Cell Line Origin PTEN status EC50
[たM]
AKT inhibitor mTOR inhibitor
BPH-1 Benign prostate + >50 5.8 (3.4-8.3)
P4E6Localised prostate
tumour+ >50 10.2 (7.2-13.2)
LNCaPLymph node
metastasis- 0.4 (0.3-0.5) 1.03 (0.78-1.28)
PC3 Bone metastasis - 11.0 (6.2-15.8) 0.6 (0.3-0.9)
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Treatment with AZD7328 and KU-0063794
reduces levels of phospho-biomarkers and
induces cell cycle arrest in LNCaP cells
In order to validate the speciicity of the inhibitors on the PI3K/AKT/mTOR pathway, we then determined
the activity of selected phospho-biomarkers, by western
blotting. PTEN-positive (BPH1) and PTEN-negative
(LNCaP and PC3) cell lines were treated with 1 たM AZD7328 and KU-0063794 for 2 hours. The results
showed a reduction in phospho-PRAS40 (T246), which is
a substrate of AKT kinase, in all the cell lines, following
treatment with AZD7328 (Figure 2A). However, phospho-
FOXO (T24/T32) levels decreased only in LNCaP cells.
Figure 1: Cell viability decreases following inhibition of AKT and mTOR in patient-derived prostate cultures. (A)
Primary cultures derived from cancers (n=5) and normal prostate (n=1) were treated with either 3 たM AZD7328, 3 たM KU-0063794 or a combination of 3 たM AZD7328 + 3 たM KU-0063794 in triplicate for 24 hours and 48 hours. Following treatment, the cells were harvested, stained with trypan blue and counted. The percentage of viable cells was calculated and normalized to the vehicle control (0.04% DMSO).
Signiicant differences (p value < 0.05) in cell viability are indicated on the graphs. (B) Primary prostate cancer samples H252/12, H163/12,
H149/12, and H135/11 were treated with 3 たM AZD7328, 3 たM KU-0063297 or a combination of 3 たM AZD7328 + 3 たM KU-0063794 for 72 hours. Following treatment, the cells were harvested, sorted into committed basal (CB), transit amplifying (TA) and stem-like cells
(SC) and counted using trypan blue exclusion. Percentage of viable cells was calculated and normalized relative to the vehicle control
(0.06% DMSO). Error bars represent the standard deviation. Signiicant difference (p <0.05) in cell viability was only observed in stem cell fraction treated with the combination of 3 たM AZD7328 + 3 たM KU-0063794 in comparison to vehicle control (indicated on the graph). (C) Cell cycle distribution remains unchanged in primary prostate cultures after treatment with AZD7328 and KU-0063794. Five prostate
cultures (2 BPH and 3 cancers) were treated with 5 たM AZD7328, 5 たM KU-0063794 or 5 たM AZD7328 + 5 たM KU-0063794 for 72 hours. Following treatment, non-adherent cells were removed and remaining cells were harvested, ixed with 70% ice-cold ethanol, and stained with propidium iodide and analysed by low cytometry. Control cells were treated with 0.1% DMSO. The signal was recorded in the PE channel and debris (subG1 phase) were excluded from the analysis.
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Treatment with KU-0063794 resulted in a signiicant reduction of phospho-S6 (S235/236) (mTORC1 substrate)
and phospho-AKT (S473) (mTORC2 substrate) in all
three cell lines. This result conirmed the speciicity of the inhibitors and their eficacy at a concentration of 1 たM, especially in LNCaP cells, where reduction of all respective biomarkers was observed.
Next, the effect of AKT or mTOR inhibition on
cell cycle distribution was analysed. The results showed
that the malignant LNCaP cells were the most affected.
The majority of cells (86%) arrested in G0/G1 following
72-hour treatment with 1 たM KU-0063794, and 81% after treatment with 1 たM AZD7328, compared to 65% for the vehicle control (Figure 2B). Cell cycle distribution
in BPH1 and PC3 cells did not change signiicantly after treatment with either of the inhibitors.
Treatment of patient-derived cultures with
AZD7328 induces autophagy and an increase in
Phospho-ERK1/2 levels
As there was more variability in primary cultures�
response to low concentrations of the inhibitors, we next
investigated whether treatment with higher concentrations
(10 – 50 たM) would result in cell death of the patient-derived cultures. A 72-hour treatment resulted in dramatic
morphological changes, which were very speciic for each inhibitor (Figure 3A). Treatment with 25 たM AZD7328 increased vacuolation of a cancer culture (H282/12),
indicative of autophagy (Figure 3A, middle panel). In
contrast, treatment of the same cells with 25 たM KU-0063794, caused membrane blebbing in a number of
cells, indicative of apoptosis (Figure 3A, right panel). To
Table 2: Primers used for PTEN mutational and LOH analysis.
Primer sequences/ Analysis type Forward Reverse
Exon 5 sequencing (mutational
analysis)5� ACCTGTTAAGTTTGTATGCAAC 3� 5� TCCAGGAAGAGGAAAGGAAA 3�
LOH intron 2 (AFMa086wg9) 5� AAATGTACGGTTCATTGACTT 3� 5� GACTGACTACAAATGGGCA 3�
LOH intron 8 5� CATTCTTCATACCAGGACCAG 3� 5� TCATGTTACTGCTACGTAAAC 3�
Figure 2: Inhibition of AKT and mTOR kinase reduces phospho-biomarker expression and induces cell cycle arrest in LNCaP cells. (A) Prostate cell lines BPH1, LNCaP and PC3 were treated with either 1 µM AZD7328 or 1 µM KU-0063794 for
2 hours to measure acute response in change of biomarkers phosphorylation status. Following treatment, cells were lysed on ice, spun
down and supernatant was collected. 20 µg of protein was loaded and run on 4-12% Bis-Tris polyacrylamide gel. Resolved protein was
then electrotransferred to PVDF membranes and immunostained with antibodies against Phospho-AKT (S473), Phospho-FoxO1/FoxO3a
(T24/T32), Phospho-PRAS40 (T246), Phospho-S6 (S235/236), total AKT, FoxO3a, total S6 and Vinculin, which was used as a loading
control. Dashed line separating lane 1 (0.1% DMSO control) from the remaining 2 lanes indicates where images were cropped. (B) Cell
cycle distribution was determined in BPH1, LNCAP and PC3 cells. Cells were treated with either 1 µM AZD7328 or 1 µM KU-0063794 for
72 hours. Following treatment, cells were washed with PBS, then harvested, ixed with 70% ice-cold ethanol, and stained with propidium iodide and analysed by low cytometry. Control cells were treated with 0.1% DMSO. The signal was recorded in the PE channel, and debris were excluded from the analysis.
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follow up these observations, expression of an autophagy
marker, LC3 B was analysed by immunoluorescence. The results showed evidence of autophagosomes in the
cytoplasm in the cells treated with AZD7328, but not with
KU-0063794 (Figure 3B). Autophagy was also measured
by Western blot; conversion of LC3B-I to LC3B-II
is suggestive of autophagy, which was observed after
treatment with AZD7328 (Figure 3C and 3D). In addition,
Figure 3: Autophagy is induced and phospho-ERK1/2 levels increase in patient-derived cultures after treatment with AKT inhibitor. (A) Phase contrast images of primary cells (H282/13) from a Gleason 7 (4+3) prostate cancer, treated with 0.5% DMSO
(left), 25 たM AZD7328 (middle) and 25 たM KU-0063794 (right) for 72 hours. Increased vacuolization of AZD7328-treated cells and membrane blebbing of KU-0063794-treated cells are indicated (red arrows). Control cells were treated with 0.5% DMSO. (B) LC3 B
expression increases in primary prostate cancer cells H310/13 (GL7) following treatment with AZD7328. Expression of the autophagy
marker, an LC3 B, was determined using Immunoluorescence. The cells were seeded into collagen I-coated 8-well chamber slides and treated with either 25 たM AZD7328 or 25 たM KU-0063794 for 96 hours and then ixed in 4% PFA. Control cells were treated with 0.25% DMSO. Scale bar on the phase contrast images represents 200たm and on the luorescent images 63 たm. (C) Phospho-ERK1/2
levels increase in prostate primary cultures following treatment with AZD7328 and KU-0063794. Primary BPH (H277/13) and cancer
cells (H278/13) were treated with 5 たM AZD7328, 5 たM KU-0063794 or 5 たM AZD7328 + 5 たM KU-0063794 for 72 hours. Westerns were carried out and biomarkers detected (phospho-AKT (S473), total AKT, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2 and LC3
B). Vehicle control cells were treated with 0.1% DMSO. Staining with く-actin antibody was used as a loading control. The bands were quantiied using Image J software and the expression normalized to the vehicle control; the values are shown below the blot. (D) Primary
prostate cancer cells H282/13 were treated for 72 hours with up to 50 たM of AZD7328 and KU-0063794 or a 10 たM AZD7328 + 10 たM KU-0063794 combination of both. Westerns were carried out and biomarkers detected (phospho-AKT, total AKT, phospho-ERK1/2, total
ERK1/2, phospho-S6, total S6, cleaved PARP and LC3 B). Vehicle control cells were treated with 0.5% DMSO. Staining with く-actin antibody was used as a loading control. The bands were quantiied using Image J software and the expression normalized to く-actin; the values are shown below the blot.
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apoptosis was measured using cleaved PARP (Figure 3D).
The results also demonstrated an increase in cleaved
PARP levels in cells treated with AZD7328, but not KU-
0063794 (Figure 3D), suggesting that despite activation of
autophagy, cells died via apoptosis.
Additionally, treatment with AZD7328 caused
an increase in phospho-ERK1/2 levels in all analysed
cultures (Figure 3C and 3D), indicating an activation of
the MAPK pathway in those cells. Treatment with KU-
0063794 did not increase levels of phospho-ERK1/2 in
any of the cancer cultures. However, an increase in MAPK
pathway activity was observed in KU-0063794-treated
BPH cultures (Figure 3C).
As EGF is an activating ligand of the MAPK
pathway and is a component of primary cell culture media
used in our laboratory, we sought to determine whether
the EGF in the culture medium affected the outcome of
treatment with the inhibitors. Selected phospho-biomarker
levels were determined in H268/12 (BPH culture)
following treatment with 5 たM AZD7328, 5 たM KU-0063794 or a combination of 5 + 5. The results showed
that treatment with AZD7328 resulted in a 9-fold increase
in phospho-AKT in the presence of EGF, whereas in the
absence of EGF, a 12.1-fold increase in expression was
observed (Supplementary Figure 3). Phosphorylation of
ERK1/2 increased by 2.8-fold following treatment with
AZD7328 (in the presence of EGF) and by 1.2 in the
absence of EGF, which suggests that the ERK activation
may be mediated via signalling from growth factors,
such as EGF. However, phosphorylation of S6 remained
at a comparable level following treatment, either in the
presence or absence of EGF. We concluded that addition
of EGF to the stem cell media affected, but did not mask
the outcome of treatment. Therefore, in subsequent
experiments EGF was included in the culture medium for
primary cells.
Table 3: Patient data summary.
Patient ID Operation Diagnosis (Gleason Grade) Age Hormone Therapy
H233/12 Radical Prostatectomy Normal 61 Naive
H268/12 TURP BPH 62 -
H277/13 TURP BPH 68 -
H313/13 TURP BPH 65 -
H278/13 Radical Prostatectomy Cancer - Gleason 6 (3 + 3) 72 Naive
H315/13 Radical Prostatectomy Cancer - Gleason 6 (3 + 3) 62 Naive
H237/12 Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 66 Naive
H240/12 RA+RB Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 66 Naive
H252/12 Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 60 Naive
H310/13 R+L Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 67 Naive
H329/13 LB Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 53 Naive
H373/13 Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 82 Naive
H377/13 Radical Prostatectomy Cancer - Gleason 7 (3 + 4) 60 Naive
H233/12 Radical Prostatectomy Cancer - Gleason 7 (4 + 3) 61 Naive
H282/13 RA+RB Radical Prostatectomy Cancer - Gleason 7 (4 + 3) 62 Naive
H236/12 Radical Prostatectomy Cancer - Gleason 8 (4 +4) 61 Naive
H163/12 Channel TURP Cancer - Gleason 8 (4 +4) 65 Yes
H239/12 RB Radical Prostatectomy Cancer - Gleason 9 (4 + 5) 50 Naive
H149/12 Channel TURP Cancer - Gleason 9 (4 + 5) 78 Yes
H224/12 Channel TURP Cancer - Gleason 9 (4 + 5) 76 Yes
H271/12 Channel TURP Cancer - Gleason 9 (4 + 5) 86 Yes
H135/11 Channel TURP Cancer - Gleason 9 (5 + 4) 56 Yes
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Combined treatment with AZD7328 and
MEK1/2 inhibitors further enhances phosphorylation of ERK1/2 in patient-derived
cultures but induces senescence
To establish whether inhibition of MEK1/2 would be
suficient to overcome the compensatory effect of MAPK pathway activation, following inhibition of AZD7328,
two small molecule inhibitors of MEK1/2, AZD6244
and RO-5126766, were tested. Following conirmation of inhibitory activity of both MEK1/2 inhibitors in
prostate epithelial cultures (Supplementary Figure 4),
combination treatment with AZD7328 + AZD6244 or
AZD7328 + RO-512 were performed in patient-derived
cancer cultures, for 72 hours. The results from Western
blotting showed a further increase in phospho-ERK1/2
levels after treatment with either of the combinations
(Figure 4A and 4B), even after treatment with a high
concentration (up to 25 µM) of the MEK1/2 inhibitors.
This suggests that despite conirmed inhibitory activity against MEK1/2 kinase, neither AZD6244 nor RO-512
inhibitor is capable of preventing ERK1/2 phosphorylation
following compensatory activation of MAPK pathway.
However, despite this increase in phospho-ERK1/2
levels, the effect of combined treatment results in cellular
senescence as detected by the acidic く-galactosidase staining (Figure 4C).
Combined inhibition of AKT and mTOR
decreases tumour frequency in a patient-derived
xenograft model
To determine whether AKT and mTOR blockade
could inhibit tumour growth, tumour cells were harvested
Figure 4: Senescence is induced following combination treatment with AZD7328 and MEK1/2 inhibitors. Primary cancer
cultures were treated with a range of concentrations of AZD6244, AZD7328 or combinations of AZD6244 + AZD7328 (A) or RO-512
+ AZD7328 (B) for 72 hours. Westerns were carried out and biomarkers detected (phospho-AKT, total AKT, phospho-ERK1/2 and total
ERK1/2). Vehicle control cells were treated with 0.5% DMSO (A) or 0.2 % DMSO (B). The bands were quantiied using Image J software and the expression normalized to く-actin; the values are shown below the blot. (C) Primary BPH culture H373/13 was treated with either
1 たM AZD6244, 1 たM RO-512 or a combination of 1 たM AZD6244 + 1 たM AZD7328 or 1 たM RO-512 + 1 たM AZD7328 for 72 hours. Following treatment, cells were ixed and stained according to the manufacturer’s instructions and phase contrast images were taken. く-galactosidase – positive cells were observed (red arrows) as well as く-galactosidase negative cells (green arrows). Vehicle control cells were treated with 0.02% DMSO. Scale bar represents 400 たm.
Oncotarget9www.impactjournals.com/oncotarget
from a human prostate cancer xenograft, (derived from a
patient with hormone-naïve Gleason 7 disease), and were
subsequently treated with either 10 たM AZD7328, 10 たM KU-0063794 or a combination of 10 + 10, for 72 hours.
Following treatment, the cells were engrafted (at limiting
dilutions) into Rag2-/-けC-/-mice to determine the effect of
treatment on tumour frequency. The results showed that
xenograft tumour cells were still able to initiate tumours
following treatment with either inhibitor or a combination
of both (Table 4). However, tumour frequency was
signiicantly reduced with the combination treatment (P
=0.010) (Table 5). In contrast, neither AZD7328 nor KU-
0063794 blockade affected tumour outgrowth.
An additional effect of treatment with a combination
of the inhibitors was an 8-day increase in the tumour
latency compared to vehicle control (38 days and 30 days,
respectively). There was no difference in tumour latency
following treatment with either inhibitor alone.
DISCUSSION
The PI3K/AKT/mTOR and the Androgen Receptor
signalling pathways are important drivers of prostate
cancer growth and progression. Despite progress in
prostate cancer treatment with the development of second
generation anti-androgen agents, patient outcome remains
unsatisfactory. Following the development of endocrine
resistance, patients are typically treated with docetaxel,
which is effective in 40 � 55 % of patients [4, 35�37].
Targeting of the PI3K/AKT/mTOR pathway has been
investigated in a number of studies as an alternative
treatment for patients with over-activation of the pathway
[38, 39]. Since a reciprocal feedback between the
AR and the PI3K pathways has been demonstrated in
prostate cancer, a combination of inhibitors targeting the
PI3K pathway and anti-androgens, such as abiraterone
and enzalutamide is currently in clinical trials [8, 40].
However, targeting androgen-dependent prostate cells
does not eradicate the stem cell component of prostate
tumours, which we and others have shown to have an AR-
negative phenotype [24, 28, 41]. Therefore alternative
combinations of drugs are urgently required if we are
to progress in the treatment of prostate cancer and the
potential of PI3K pathway inhibitors has to be fully
elucidated.
The aim of this study was to determine the
importance of the PI3K/AKT/mTOR pathway in human
prostate cancer. This was performed by targeting the
pathway with AKT and mTOR inhibitors in prostate
cancer cell lines, primary cell cultures and a �near-patient�
xenograft. Activity of the small molecule inhibitors against
AKT and mTOR kinases used in this study have also been
tested in a number of other tumour types [42, 43].
The results of treatment with AZD7328 and KU-
0063794 showed that PTEN-negative LNCaP cells were
the most susceptible to treatment. Treatment of primary
cultures (derived from patients with BPH and cancer)
conirmed variability in response between patient samples, even from patients with the same Gleason grade. In
contrast to established prostate cancer cell lines, PTEN
Table 4: Tumour initiation frequency is lower in xenograft cells treated with a combination of AKT and mTOR
inhibitor.
Number of inoculated
cells
10 000 cells 1000
cells
100 cells 10 cells 1 cell Tumour initiation
frequency
(95% CI)Treatment
0.2% DMSO 2/2 2/2 2/2 1/2 0/2 16.6 (98.9-3.16)
10 µM AZD7328 2/2 2/2 2/2 0/2 0/2 43.8 (201.1-9.84)
10 µM KU-0063794 2/2 2/2 2/2 1/2 0/2 16.6 (98.9-3.16)
10 +10 Combination 2/2 2/2 0/2 0/2 0/2 434.6 (2008.4-94.36)
Table 5: Pairwise tests for differences in tumour frequencies.
Group 1 Group 2 p value
AZD7328 Combination 0.071
AZD7328 DMSO 0.443
AZD7328 KU-0063794 0.443
Combination DMSO 0.010
Combination KU-0063794 0.010
DMSO KU-0063794 1
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protein expression was observed in all primary cells
investigated, including Gleason 8 tumours. Although
there was variability in the levels of PTEN expression,
none of the cultures showed a complete loss of PTEN.
However, 3 out of 7 (43%) patient-derived cultures had
mutations in exon 5 of the PTEN gene and 2 out of 7
(28.6%) showed loss of heterozygosity in tumour DNA,
in comparison to matched lymphocyte DNA. PTEN loss
is typically observed in 70-80% of castration resistant
prostate tumours [32, 33, 44]. This discrepancy can be
explained because the majority of cultures used in this
study were derived from treatment naïve patients with
early stage disease, whereas PTEN is more frequently
lost in advanced prostate cancers. Only 12% of radical
prostatectomy samples show complete loss of PTEN
[45]. Nevertheless these cultures are relevant because it
is imperative that combinations of inhibitors that delay
progression to advanced disease, after initial androgen
deprivation therapy or radiotherapy, are identiied.Targeting the PI3K/AKT/mTOR pathway has
been proposed as a clinical strategy to eliminate cancer
stem cells. For example, treatment of anaplastic thyroid
cancer with a PI3K inhibitor, LY294002, attenuated
the self-renewal of cancer stem cells and induced cell
differentiation [46]. Similar indings were also reported in acute myeloid leukaemia and in breast cancer stem cells,
where it was shown that the PI3K/AKT/mTOR pathway
was critical for cancer stem cell proliferation and survival
[47, 48]. Moreover, treatment with a chemotherapeutic
drug cisplatin, results in a signiicant increase in breast cancer stem cells, which could be partially attributed to
the activation of the PI3K/AKT/mTOR pathway [49].
However, combined treatment with cisplatin and the
mTOR inhibitor rapamycin synergistically inhibited
cancer stem cell-mediated primary and metastatic cancer
growth [49]. These recent indings emphasise the need to test combination treatments, as a more successful
approach to targeting cancer stem cells. Our indings suggest that despite differences between patients�
responses, targeting stem cells with a combination of
AZD7328 and KU-0063794 might potentially be more
successful than a monotherapy with either drug alone.
Further investigation is needed to identify differences
between treatment responders and non-responders, in a
more eficient stratiication of patients in the clinic.Treatment with high concentrations (≥ 25 たM)
of AKT and mTOR inhibitors induced dramatic
morphological changes, such as vacuolation of cells and
membrane blebbing. Although these concentrations are
much higher than exposures achieved in man by AKT and
mTOR inhibitors of similar potency, the biology invoked
as a consequence of greater target inhibition is of interest.
Membrane blebs are spherical outgrowths of the plasma
membrane that occur upon membrane detachment from
the underlying cytoskeleton and are indicative of apoptosis
[50]. However, we observed no increase in cleaved PARP
expression after mTOR inhibitor treatment. This could be
due to a minority of cells showing apoptotic features, and
therefore being undetectable by western blotting. Increased
vacuolation of cells following treatment with the AKT
inhibitor was morphologically indicative of autophagy
and the formation of autophagosomes. Autophagy is
a cellular catabolic degradation process in response to
starvation or stress, in which cellular organelles, proteins
and cytoplasm are digested and recycled to maintain
metabolism [51, 52]. A clear increase in the conversion of
LC3-B I to LC3-B II (autophagy marker) demonstrated by
both western blotting and immunoluorescence conirmed the morphological evidence. Treatment with the ATP-
competitive AKT inhibitor (AZD7328) does induce
autophagy in human bladder cancer cell lines, which can
be overcome by treating the cells with chloroquine [53].
However, autophagy can also prevent tumourigenesis,
acting as a tumour suppressor [52, 54]. Accumulation of
the autophagy substrate p62/SQSTM1 is suficient to both activate the MAPK pathway and increase cell proliferation
in PI3K-expressing mammary 3D structures [54].
Increased cell proliferation and MAPK activation, due to
inhibition of autophagy, was only evident under 3D culture
conditions, and not in monolayer cells [54]. In 3D, the cells
treated with AZD7328 showed the expected increase in
autophagy, as well as activation of the MAPK pathway.
We observed activation of the MAPK pathway
following treatment with AZD7328, and so the primary
cultures were also treated with a combination of AZD7328
and MEK1/2 inhibitors to determine the outcome of
inhibiting this compensatory pathway. Surprisingly, a
combined treatment with AZD7328 and either AZD6244
or RO-512 resulted in an increased phosphorylation
of ERK1/2 to even greater levels than treatment with
AZD7328 alone. Davies and colleagues have previously
demonstrated activity and eficiency of the AZD6244 in a large panel of human cancer cell lines as well as in
xenografts derived from them [55]. It has been reported
that a majority of cell lines with a mutation in KRAS,
NRAS or BRAF genes showed a signiicantly increased sensitivity to MEK inhibition, resulting in cell growth
inhibition and induction of apoptosis [55]. In the human
colon carcinoma xenograft HCT-116, a combination
treatment of the AZD6244 and docetaxel resulted in
enhanced tumour growth inhibition, in comparison to
monotherapy [56]. However, the rate of mutations in Ras/
MEK/ERK pathway in clinical prostate cancer is very low;
only up to 7% of clinical samples have activating point
mutations of Ras and around 4 % are positive for BRAF,
the most common activating mutation of RAF [57].
Similarly, eficacy of RO-512 has been demonstrated in a number of human cancer cell lines and also in the HCT-
116 xenograft model [58]. However, in our patient-derived
cultures, neither of the MEK1/2 inhibitors was able to
overcome the compensatory ERK1/2 activation caused by
inhibition of AKT.
Oncotarget11www.impactjournals.com/oncotarget
It is unclear why combination treatment of prostate
cancer primary cells with AZD7328 and MEK1/2 inhibitors
did not reduce ERK1/2 phosphorylation. A possible
explanation is that we could be observing a profound stress
response where other MAPKs are activated and although
phospho-ERK1/2 antibody we used does not cross-react with
the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases, this phenomenon will have to
be investigated further. However, acidic く-galactosidase staining of treated cultures revealed that the cells became
senescent after the combination treatment. Senescence is
frequently triggered in tumours in response to chemotherapy
and radiotherapy treatment in cancer patients [59, 60] and
it has been demonstrated that PTEN-loss-induced cellular
senescence inhibits tumorigenesis in vivo in a human xenograft
model of prostate cancer [61]. Induction of senescence in
cancer cells could indeed be a good therapeutic strategy for
treatment of prostate cancer if it established stable disease [61].
The PI3K/AKT/mTOR and the Ras/MEK/ERK
pathways are the two major hyper-activated pathways, which
promote cell proliferation, survival and metastasis in human
cancer [11, 62]. Signiicant activation of MAPK signalling has been observed in both primary and in metastatic cancer
[63]. Activation of the MAPK pathway has been implicated
in resistance to the PI3K/AKT/mTOR signalling inhibition in
other studies [64]. Here, we present for the irst time, evidence that the MAPK pathway acts as a compensatory pathway
in primary prostate epithelial cell cultures following AKT
inhibition. However, the possibility of a third compensatory
pathway cannot be discounted by our data.
Despite our observation that the Ras/MEK/ERK
compensatory pathway is activated in primary cells
following the PI3K/AKT/mTOR inhibition, the ex vivo
treatment of the PDX with a combination of KU-0063794
(mTOR inhibitor) and AZD7328 (AKT inhibitor) showed
an effective reduction in tumour outgrowth. Therefore
this combination treatment alongside anti-androgen
therapy has the potential to be more effective than single
treatments. However, due to the compensatory pathway
and the further possibility of as yet unknown effects, the
outcome of drug combinations may not be predictable and
may have to be determined on a patient speciic basis. The utilisation of primary cell cultures from multiple individual
patients will allow elucidation of mechanisms of action of
drugs, mechanisms of resistance and variation in patient
response, at a level of which cannot be predicted using the
standard panel of available prostate cancer cell lines.
MATERIALS AND METHODS
Inhibitors
The AKT inhibitor AZ7328, mTOR inhibitor KU-
0063794 and MEK inhibitor Selumetinib (AZD6244;
ARRY-142886) were obtained from AstraZeneca, and the
MEK1/2 inhibitor RO-5126766 (RO-512) was purchased
from MedKoo Biosciences, Inc., NC, USA. AZ7328 is
an ATP-competitive, selective inhibitor of AKT [53] that
inhibits all three AKT isoforms with an IC50 of < 50 nM in
isolated enzyme assays, and inhibits the phosphorylation
of GSK3く in PTEN null MDA-MB-468 breast cancer cells with an IC50 of 91 nM. KU-0063794 is a highly
selective inhibitor of mTOR kinase [43] and Selumetinib
is a selective, non-ATP competitive inhibitor of MEK1/2
kinases [55]. RO-5126766 is an allosteric MEK inhibitor
which has been shown to additionally supress feedback
induction of RAF-dependent MEK phosphorylation [58].
Culture of cell lines
Human prostate cell lines used in the experiments
included: BPH1, P4E6, LNCaP and PC3 cells. LNCaP and
PC3 cells were purchased from American Type Culture
Collection (ATCC), BPH1 were a gift from Dr Hayward
(NorthShore University HealthSystem Research Institute),
and P4E6 cells were derived in our laboratory [65]. BPH1
and LNCaP cells were cultured in Roswell Park Memorial
Institute-1640 (RPMI) (Invitrogen Ltd) supplemented with
5% or 10% Foetal Calf Serum (FCS) (PAA), respectively,
and 2 mM L-Glutamine (Invitrogen Ltd). PC3 cells were
cultured in Ham�s F-12 medium (Lonza) supplemented
with 7% FCS and 2 mM L-Glutamine. P4E6 cells were
cultured in Keratinocyte Serum-Free Medium (KSFM)
(Invitrogen Ltd) supplemented with 2% FCS, 2 mM
L-Glutamine, 5 ng/ml Epidermal Growth Factor (EGF),
50 たg/ml bovine pituitary extract (Invitrogen Ltd). All cell lines were cultured in T25 or T75 cell culture lasks (Corning) at 37°C in 5% CO
2.
Tissue collection and culture of tumour cells
Prostate tissue was obtained from patients
undergoing radical prostatectomy or transurethral
resection of the prostate (TURP) (Table 3), with informed
patient consent and approval from the local Research
Ethics Committee (07/H1304/121). All patient samples
were anonymized. Epithelial cultures were prepared as
described previously [31]. Cell cultures were maintained
in stem cell media (SCM), which consisted of keratinocyte
growth medium supplemented with 5 ng/ml Epidermal
Growth Factor (EGF), 50 たg/ml bovine pituitary extract (Invitrogen Ltd), 2 ng/ml stem cell factor (First Link), 100
ng/ml cholera toxin (Sigma-Aldrich Company Ltd) and 1
ng/ml GM-SCF (First Link). Epithelial cells were cultured
on collagen I � coated plates in the presence of irradiated
(60 Gy) STO (mouse embryonic ibroblasts) cells.
Generation of xenografts and isolation of tumour
cells for ex-vivo treatment
All animal work was approved by the University
of York Animal Procedures and Ethics Committee
Oncotarget12www.impactjournals.com/oncotarget
and performed under a United Kingdom Home Ofice License. Human prostate cancer tissue was obtained from
a patient with hormone naïve, Gleason 7 disease. Tumour
Xenografts were derived according to the method outlined
in Kroon et al., 2013 [66]. Xenografts were maintained
subcutaneously in BALB/c/Rag2-/-けC-/- mice, which are
highly receptive for engrafting of human tissue due to
deiciencies in T, B and NK cell development [67].15 mm3 tumours were harvested from humanely
euthanized mice and human Lin-/CD31-, were isolated
according to Kroon et al., 2013 [66]. Tumour cells
were resuspended in D10 medium, plated onto collagen
I � dishes and treated for up to 72 hours with AKT and
mTOR inhibitors. Following treatment, the cells were
resuspended in Matrigel and injected subcutaneously into
the lanks of a Rag2-/-けC-/-, 2x105 irradiated STO were used
as carrier cells. Mice were monitored weekly for tumour
growth. Once the tumours were detectable they were
measured every 2-3 days using a digital calliper (Duratool
DC150). Tumour volume (mm3) was calculated using the
ellipsoidal formula: (length x width2)/2. Tumour initiation
frequency was calculated using Extreme Limiting Dilution
Analysis software available online at (http://bioinf.wehi.
edu.au/software/elda/), which provided an estimate of
frequency with 95% conidence intervals and includes the Pearson�s chi-square test for pairwise analysis to
determine differences between treatment groups.
Treatment of subpopulations of primary cell
cultures
Primary cells were seeded onto collagen I-coated
10 cm dishes, 24 hours prior to treatment. Cells were
treated with increasing concentrations (0.1 たM to 50 たM) of AZD7328, KU-0063794 or MEK1/2 inhibitors either alone or in combination for up to 72 hours. Control
cells were treated with DMSO. Following treatment,
g2く
1
hi/CD133+(stem-like cells), g2く
1
hi/CD133- (transit
amplifying, TA) and g2く
1
low (committed basal, CB)
cells were isolated by MACS (Miltenyi Biotec Ltd.), as
described previously [68, 69]. Briely, cell subpopulations were enriched and selected from primary cell cultures,
irst using collagen adherence to separate g2く
1
hi and g2く
1
low
cells. This was followed by incubating the g2く
1
hi cells with
anti-human CD133 microbeads and selecting CD133+
cells using MACS magnetic bead selection (Miltenyi
Biotec Ltd). This results in three cell subpopulations �
g2く
1
hi/CD133+(stem-like cells), g2く
1
hi/CD133- (transit
amplifying, TA) and g2く
1
low (committed basal, CB) cells.
PTEN mutational analysis and loss of
heterozygosity (LOH) analysis
The Polymerase chain reaction (PCR) was used to
amplify matched lymphocyte and tumour DNA from the
patient primary cultures to determine mutational status
and loss of heterozygosity of the PTEN gene. Standard
PCR conditions were adapted for the analysis of PTEN
exon 5 [34]. PCR products were column puriied using Qiagen Quick PCR clean up kit (Qiagen, UK) and then
directly sequenced using PTEN exon 5 sequencing primers
(Table 2). The presence of mutations was determined by
using EditSeq software (DNASTAR).
Loss of heterozygosity in PTEN was assessed
using two LOH probes described in Dahia et al
[34]; they included a dinucleotide repeat in PTEN
intron 2 (AFMa086wg9) (probe 1) and a G/T sequence
polymorphism in PTEN intron 8 (probe 2). The reverse
primer for intron 2 was labelled with a JOE luorescent dye enabling PCR fragment analysis, which was
performed using Applied Biosystems ABI3130XL
and data analysed using Gene Mapper software (Life
Technologies). LOH was identiied in the heterozygous tumour samples as a reduction in one of the peaks, which
corresponded to one of the alleles of the PTEN gene. The
G/T polymorphism was analysed using digestion with
restriction enzyme HincII (New England Biosciences),
where the polymorphism is differentially cleaved in
heterozygous and homozygous individuals.
Agarose gel electrophoresis
PCR products were visualised using 1.5% agarose
gel electrophoresis prepared with Tris-Acetate-EDTA
buffer and run at 80V. The products of the HincII digestion
reaction were separated in a 3% agarose gel in Tris-Borate-
EDTA buffer and run at 100V.
SDS-PAGE and Western blotting
Adherent and non-adherent cells were harvested
and resuspended in Cytobuster protein extraction reagent
(Novagen) supplemented with the addition of protease
inhibitor cocktail (Roche) and phosphatase inhibitor
cocktail (Roche). Whole cell protein lysates were
quantiied using the Bicinchoninic acid assay (BCA) protein assay kit (Thermo Scientiic), according to manufacturer�s instructions.
20 たg of total cell lysate was heated to 100°C for 10 min in sample buffer (4 x SDS loading buffer
(10% (v/v) glycerol, 62.5 mM Tris-HCl pH 6.8, 1%
(w/v) SDS, 65 mM DTT and bromophenol blue),
and subjected to PAGE and electrotransfer onto
Immobilon-P membrane (Millipore). Membranes were
blocked for 1 hr with 10% blocking reagent (Roche)
in TBS-Tween, followed by incubation with primary
antibodies at 4°C for 2 hours or overnight. Antibodies
were purchased from Cell Signaling Technology, unless
otherwise speciied, and included: AKT (pan) (#4691), phospho-AKT (Thr308) (#2965), phospho-AKT (Ser473) (#4060), 4E-BP1 (#9452), phospho-4E-BP1 (Thr37/46) (#2855), p44/42 MAPK (ERK1/2) (#4695),
Oncotarget13www.impactjournals.com/oncotarget
phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)
(#4370), LC3-B (abcam, ab51520), PRAS40 (#2691), phospho-PRAS40 (Thr246) (#2997), PTEN (#9188), S6 Ribosomal Protein (2217), phospho-S6 (Ser235/236)
(#2211), FoxO3a (#9467), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) (#9464), Vinculin (Sigma V9131), く-actin (Sigma, A4700). Chemiluminescent detection was performed using the BM Chemiluminescence
Blotting Substrate (POD) system (Roche). Labelled
proteins were visualised using ECL Hyperilm (Amersham) and manually processed using Kodak GBX
developer and ixer solutions (SLS).
Flow cytometry and cell cycle analysis
Cell lines and primary prostate cells were used for
cell cycle analysis following treatment. Cells were ixed in ice-cold 70% Ethanol, then incubated on ice for 30
minutes and resuspended in PBS. RNase A (at 1 mg/ml
inal concentration) and propidium iodide (PI; 400 µg/ml inal concentration) were added, and cells incubated at 37°C for 30 min. Cells were then placed on ice, analysed
on a CyAn ADP low cytometer (Dako Cytomation) and PI luorescence was recorded in the PE channel. To prevent bleaching of the luorescence, samples were protected from light where possible during the
procedure. The results were analysed using the Summit
v4.3 software.
Immunoluorescence
Primary prostate cells were seeded at a density of
7.5 x 104 per well in BD-Biocoat collagen I-coated 8-well
chamber slides and incubated at 37°C at 5% CO2 for 24
hours before treatment. Following treatment, cells were
ixed, permeabilised and stained as described previously [69]. Antibodies used were LC3-B (abcam, ab51520
at 1:2000) and secondary anti-rabbit Alexa Fluor 488
(Invitrogen, A-11034 at 1:300). Slides were mounted in
Vectashield containing DAPI (Vector laboratories). Images
were captured using a Nikon Eclipse TE300 luorescent microscope (Nikon). Secondary only antibodies, where
the primary antibody was replaced with PBS, were used
as controls.
く-Galactosidase staining assay
Cells were seeded onto 10 cm dishes and, following
treatment, an acidic く-Galactosidase assay (Cell Signaling Technology) was performed as an indicator of
senescence. Briely, cells were washed once with PBS and ixed for 15 minutes at room temperature, washed twice with PBS, and then incubated with く-Galactosidase staining solution, at pH 6.1, overnight at 37°C in a dry
incubator. The staining results were analysed using an
Evos XL transmitted light microscope (AMG) at 10x and
20x magniication.
ACKNOWLEDGEMENTS
We thank the urology surgeons and patients from
Castle Hill Hospital for kind donations of clinical prostate
samples. We would also like to thank Paul Berry for the
assistance with the in vivo work.
CONFLICTS OF INTEREST
Barry Davies is an employee and shareholder
of AstraZeneca. No potential conlicts of interest was disclosed by the other authors.
GRANT SUPPORT
This work was funded by BBSRC and AstraZeneca
with the support from Yorkshire Cancer Research.
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